The most aggressive form of brain malignancies, glioblastoma multiforme (GBM) represents a particularly acute unmet medical need. GBM are highly invasive and do not respond to immune checkpoint inhibition due to their suppressive microenvironment. Oncolytic viruses engineered to selectively replicate and kill malignant GBM cells and to express transgenes that stimulate antitumor immunity are attractive approaches to improve patient outcomes as monotherapy and in combination with anti-PD-1 therapeutics. Preliminary data achieved with a prototypical oncolytic herpes simplex virus (oHSV) armed with ULBP3, a ligand for KLRK1 a stimulatory receptor expressed in CD8 and NK cells and featuring the improvements in oncolytic potency, micro-RNA attenuation and payload capacity designed by Oncorus demonstrated a significant survival benefit after a single intratumoral injection in a genetically inducible model of GBM designed to recapitulate the major genetic alterations and pathobiology of GBM. Moreover, injection of oHSV expressing ULBP3 in one tumor mass resulted in an abscopal anti-tumor response in the uninjected tumor mass. This result was surprising since the human-specific ULBP3 gene product does not recognize the mouse KLRK1 receptor and primarily acts through the recruitment of macrophages and microglia, suggesting a novel receptor for ULBP3 on these cells both in mouse and human. Preliminary studies identified tissue factor, a transmembrane receptor with a role in coagulation and signaling in GBM as this new receptor. We aim to investigate the mode of action of ULBP3 in GBM models and confirm this novel interaction. We propose to further exploit these initial findings for the design of an optimized armed oHSV for the treatment of GBM. As a first step, we will tailor the unique safety features of Oncorus oHSV, the selection of micro-RNA highly expressed in healthy brain tissues and lost in GBM to insert complementary micro-RNA target sequences in multiple genes essential to HSV replication, thus restraining viral replication to malignant cells. MicroRNA 124, 128 and 137 that showed strong differential expression are likely candidates. Additionally, payloads, including IL-12, and PD-1 and CD47 antagonists, that have been validated in GBM preclinical models, and that may complement the activity of ULBP3 will be tested stepwise in GBM cell lines and tumor cells for oncolytic activity, expression and in vivo efficacy in syngeneic GBM models and in state-of-the-art inducible genetic models. From these data the optimized combination of payloads will be introduced into our oHSV in order to create the most potent vector and clinical candidate for future human trials. !